Publications referred to by reference numbering in this specification correspond to the reference list at the end of the specification are hereby incorporated by reference in their entirety.
Control over protein synthesis rates is involved in the regulation of most biological processes and is believed to be the primary cause of numerous diseases. Regulation of the synthesis rates of biomolecules in living systems is one of the most fundamental features of biochemical and physiologic control. For this reason, measurement of biosynthetic rates in vivo has been the subject of enormous research effort over the past 50 years. Among the macromolecules that have been studied, proteins have received perhaps the most intense attention due to their central role in controlling biological processes. The measurement of protein synthesis, as for all other biomolecules, has traditionally required the use of isotopic labels (stable isotopes or radioisotopes). Many studies have described isotopic studies of protein biosynthesis (see Waterlow, 1978, and Hellerstein & Neese, 1999).
In essence, four general approaches have been described for measuring protein biosynthetic rates (Waterlow, 1979). These are: (1) exogenous labeling of proteins of interest, with subsequent re-introduction into the biological system followed by measurement of die-away curves of the labeled protein; (2) endogenous pulse-labeling of proteins from a labeled biosynthetic precursor, followed by measurement of die-away curves of the labeled proteins of interest; (3) endogenous pulse-labeling of proteins from a labeled biosynthetic precursor, followed by measurement of label incorporation curves into the proteins of interest, and comparison to estimates of the changing content of label present over time in the biosynthetic precursor pool; (4) endogenous labeling of proteins by continuous administration of a labeled biosynthetic precursor, with measurement of label incorporation into the proteins of interest, and comparison to steady-state label content in the biosynthetic precursor pool (use of precursor-product relationship).
Among these general labeling strategies, perhaps the most reliable technically and operationally is the continuous administration of a labeled biosynthetic precursor (approach #4). This approach takes advantage of a mathematical principle known as the precursor-product relationship or, in physics, Newton's cooling equation.
The conceptual basis of the precursor-product relationship is shown in FIG. 1. The central principle is that the label content of the product approaches a known value, or asymptote, which in turn is determined by and measurable as the label content in the biosynthetic precursor pool.
As summarized by Waterlow et al (1979), this use of the precursor-product relationship presents several key practical advantages compared to alternative strategies, particularly when the half-lives of the product pool molecules (e.g., proteins) are longer than the half-lives of the precursor pool molecules (e.g., free amino acids). The first advantage is that if the isotopic enrichment of the amino acid biosynthetic precursor pool can be determined and is relatively stable during the continuous label administration period, only a single time point of the protein end-product is, in principle, required to characterize the synthesis rate of the protein molecule. This is so because the basic precursor-product equation can be used in its integrated form when the precursor pool enrichment (SA) is held steady:dSB/dt=k(SA−SB).If SA is constant, SB(t)=SA(1−e−kt)or, SB(t)=k[∫0tSAdt−∫0tSB(dt)]This relationship is depicted graphically in FIG. 1.
Accordingly, multiple sampling of the protein is not required (unlike decay curves after endogenous or exogenous labeling) and multiple sampling of the precursor pool is not required (unlike pulse-labeling approaches). By maintaining a constant or near-constant isotope enrichment in the precursor pool, problems related to non-steady state corrections, non-homogeneity or incomplete mixing in the amino acid precursor pool are also avoided.
Waterlow et al (1979) showed mathematically that synthesis rates are rigorously calculable by this approach even when the protein mass is increasing or decreasing (i.e., if there is a non-steady state in the end-product pool). This feature allows for broad application of this approach, regardless of the physiologic conditions present in the system being studied.
There are some practical disadvantages of the continuous administration approach, however. The most important of these are: (1) the need for continuous administration of the isotopically labeled biosynthetic precursor, in order to maintain relative steady isotopic enrichments in the precursor amino acid pool. This requirement typically necessitates continuous intravenous infusion or frequent repeated oral dosing over many days, or even longer. The need for intravenous administration severely constrains routine medical diagnostic or field use of this approach; (2) the potentially high cost of maintaining a constant level of label in the biosynthetic precursor pool for a relatively long period of time; (3) the need to measure the interim isotopic enrichment of the biosynthetic precursor pool and establish its constancy, and (4) problems in identifying the “true precursor” pool for protein biosynthesis in living cells and individuals.
The problem of identifying the true precursor pool for biosynthesis applies to all applications of the precursor-product relationship, not just for protein synthesis, and derives from the central principle of the technique: the assumption that the labeling curve in the product approaches a known asymptote, or plateau value, which is determined by the label content of the precursor pool (FIG. 1). It is therefore essential to establish during any labeling study the actual asymptotic or plateau value that is being approached. This asymptote value can either be established by waiting long enough to allow the complete shape of the labeling curve in the product molecule to become apparent (FIG. 1) or by using a surrogate measure based upon the known biochemical organization of the protein biosynthetic system (i.e., from the label content in the free amino acid pool leading to protein synthesis). However, the biochemical organization of protein synthesis is extremely complex and unpredictable, making the latter approach subject to significant systematic errors (Waterlow 1979; Airhart 1974; Khairallah and Mortimore 1976).
Alternatively, allowing the shape of the curve to become apparent requires continuous administration for several half-lives of the protein end-product. This requirement is most often not practical, in that protein half-lives may be several days, weeks or months. It is not practical to maintain an intravenous infusion for more than 24 to 48 hours (even intravenous infusions of this length require medical personnel and monitoring) and oral administration of precursor metabolites cannot achieve stable values in metabolic pools.
Accordingly, it has long been recognized in the field (Waterlow 1979; Hellerstein and Neese 1992; Hellerstein and Neese 1999) that an ideal method would allow constant isotope levels in the precursor pool to be maintained for prolonged periods of time in a simple, non-demanding manner, for example, on the order of a few half-lives of long-lived proteins. However, there has not been a technique that has fulfilled this objective. A method for measuring protein synthesis that is widely applicable, reliable, easy to perform, inexpensive, without toxicities or complications, applicable in human subjects, free of the need for medical supervision or in-patient procedures (such as intravenous infusions), does not require complex instructions, and possesses the advantages of simple interpretation, therefore would be extremely valuable and useful in fields ranging from medical diagnostics to drug discovery, genetics, functional genomics, and basic research.